Brain biopsy and pathological diagnosis for drug‐associated progressive multifocal leukoencephalopathy (PML) with inflammatory reactions

Abstract

Progressive multifocal leukoencephalopathy (PML) is a demyelinating disease of the central nervous system caused by JC virus (JCV) infection. Although recognized as an AIDS complication in the 1980s, PML has emerged as a serious adverse event of immunosuppressive therapies since 2005, particularly disease‐modifying drugs (DMDs) for multiple sclerosis (MS). PML can also occur in patients with collagenous diseases receiving steroid therapy or with age‐related immunosuppression. In some cases, the etiology of immunosuppression remains unclear. These cases often present with early manifestations of PML, which, while common, are less well recognized, as PML was identified at more advanced stages in AIDS‐related cases. Early diagnosis poses difficulty due to unfamiliar magnetic resonance (MR) images and low viral loads in cerebrospinal fluid (CSF), and brain biopsy may be conducted. This review summarizes the PML pathology identified through biopsy. Early cytopathological changes of JCV‐infected cells, with the importance of dot‐shaped inclusions associated with promyelocytic leukemia nuclear bodies (PML‐NBs), are described. The variability of host immune responses, including PML immune reconstitution inflammatory syndrome (PML‐IRIS), is addressed. The potential role of immune checkpoint inhibitors (ICIs), such as pembrolizumab, is also explored. Understanding the pathology of early PML helps to optimize diagnostic strategies and therapeutic interventions, ultimately improving prognosis.

Abbreviations

ART

antiretroviral therapy

CNS

central nervous system

CSF

cerebrospinal fluid

DMDs

disease‐modifying drugs

ICIs

immune checkpoint inhibitors

ISH

In situ hybridization

JCV

JC virus

MS

multiple sclerosis

PCR

polymerase chain reaction

PML

Progressive multifocal leukoencephalopathy

PML‐IRIS

PML immune reconstitution inflammatory syndrome

PML‐NBs

promyelocytic leukemia nuclear bodies

INTRODUCTION

Progressive multifocal leukoencephalopathy (PML) is a disease caused by the JC virus, affecting the central nervous system (CNS). The JC virus typically infects individuals without symptoms but can reactivate when the host's immune system is compromised. ¹ , ² In the 1980s, PML became widely recognized as a complication of AIDS, characterized by rapid disease progression and poor prognosis. Magnetic resonance (MR) imaging of PML often reveals multiple demyelinated lesions, and the presence of the JC viral genome in cerebrospinal fluid (CSF) can be confirmed through polymerase chain reaction (PCR) testing, leading to a definitive diagnosis. The pathology was typically confirmed through autopsy.

In 2005, the occurrence of PML was reported in patients with multiple sclerosis (MS) who were treated with disease‐modifying drugs (DMDs). ³ , ⁴ , ⁵ Since then, PML has been recognized as a significant adverse effect associated with certain immunosuppressive or immunomodulatory drugs. ⁶ , ⁷ , ⁸ , ⁹ However, unlike AIDS‐related PML, drug‐associated PML appears to be potentially treatable, often presenting with a lower JC viral load. Disease progression tends to be slower, and there may be some residual immune function in the host. The features are commonly observed in PML with age‐related immune suppression and PML with an unknown cause of immunosuppression. Early diagnosis is crucial, yet MR imaging may not always be straightforward due to less pronounced pathological changes in the early stages. Additionally, the JC viral genome may not be detectable in the CSF due to low viral copy numbers, but a negative result does not rule out PML entirely. ¹⁰ In such cases, a brain biopsy may be necessary to establish a diagnosis. However, the tissue sample is often small and may not contain characteristic JC virus‐infected cells with enlarged nuclei. Furthermore, inflammatory reactions are relatively common, and differential diagnoses such as malignant lymphoma, vasculitis, and other inflammatory demyelinating diseases should be ruled out.

This review summarizes the features of PML in the early stage, frequently seen via brain biopsy. Mamy cases lacked typical JCV‐infected cells and exhibited inflammatory reactions. ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ The underlying pathophysiology will be discussed.

BRAIN BIOPSY AND PATHOLOGICAL DIAGNOSIS OF PML

In 2013, the American Academy of Neurology (AAN) proposed a consensus diagnostic criterion for PML. ¹⁹ According to these guidelines, when a pathological investigation is performed, the presence of a histopathological triad, comprising demyelination, bizarre astrocytes, and oligodendrocytes with enlarged nuclei, is considered diagnostic. ¹⁹ , ²⁰ However, unlike autopsies that allow for a comprehensive examination of the entire brain, biopsy samples are typically small, and the triad may not be observed (Figure 1a,c). The diagnostic criteria for brain biopsy cases are not yet established with accumulated pathological findings. To determine the diagnosis, the target sites of biopsy and the knowledge of the PML pathology in the early stage are essential. Therefore, selecting the appropriate biopsy site and thoroughly understanding early‐stage PML pathology is critical for accurate diagnosis.

Figure 1.

Pathological features of PML (progressive multifocal leukoencephalopathy) and the three‐step hypothesis. (a) A section of an autopsy brain showing an advanced PML lesion. The characteristic neuropathology, with extensively demyelinated lesions in a fused pattern, closely correlates with typical MR images of PML. (b) A schematic illustration of the progression of demyelinated lesions. The development of PML lesions is hypothesized to occur in three steps: Initiation, Extension, and Fusion. These stages are represented by green circles, black arrows, and red circles (a) and (b). For (a) and (b), the direction of lesion extension is based on the longitudinal MR images (Ono et al., Neuropathology, 2019). (c) Brain tissue samples obtained via biopsy. The samples are approximately 2 mm in size, and the diagnostic difficulties posed by these samples are significantly greater than those from autopsy. (d) Macrophages appear in the demyelinated lesions. HE staining. (e) Macrophages contain myelin particles in the cytoplasm. KB staining. (f) M2 macrophages are labeled with CD163. Immunostaining. For (d), (e), and (f), see the text, "Evaluation of demyelinating lesions in biopsy samples".

The biopsy target sites

The mechanism of viral proliferation within the affected brain was investigated, with longitudinal MR images compared with the post‐mortem brain in an autopsy case. ²¹ PML lesions likely progress through three steps and the three‐step hypothesis was proposed: initiation, extension/expansion, and fusion (Figure 1a,b). The first step, initiation, involves the emergence of small foci of viral proliferation, typically at the cortico‐medullary junction within the frontotemporal lobe. The JC virus reaches the brain via the bloodstream, forming these initial foci. The second step, extension, is characterized by the discontinuous spread of these small foci along nerve fibers, which may eventually fuse deep within the white matter where nerve fibers intersect. The third step, fusion, occurs when these proliferating viral foci converge, leading to significant tissue damage. Consequently, the development of PML lesions is dependent on neural tracts.

MR images can be categorized into four distinct patterns: A, B, C, and D, based on the initial sites of viral proliferation and the pathways of lesion extension. ²² Pattern A involves cerebral lesions affecting the precentral and frontal gyri, Pattern B involves vertical extension into deep gray matter and the brainstem, Pattern C involves infratentorial lesions affecting the cerebellum and brainstem, and Pattern D is characterized by deep white matter lesions with punctate MRI patterns (Figure 2). These four MRI patterns are consistently seen across different causes of immunosuppression and are commonly observed throughout the clinical course. For example, the MR images of a reported PML case under dimethyl fumarate treatment can be explained by a combination of patterns B and C. ²³

Figure 2.

Four representative MRI patterns of PML (progressive multifocal leukoencephalopathy) development. The extension of PML lesions is influenced by neuronal tracts, allowing MR images of PML progression to be categorized based on the initial sites and the pathways of lesion spread. Four representative patterns have been identified: A, B, C, and D. (a) A schematic illustration of the four MRI patterns. (b) The MRI image of Pattern A: cerebral lesions affecting the precentral and frontal gyri. (c) The MRI image of Pattern B: vertical lesions involving the deep gray matter and brainstem. (d) The MRI image of Pattern C: infratentorial lesions affecting the cerebellum and brainstem. (e) The MRI image of Pattern D: punctate signals in the deep white matter. For Patterns A, B and C, the direction of lesion extension is based on the longitudinal MR images. In detail, see original articles (Ono D et al., Neuropathology, 2019; Ishii J, Shishido‐Hara Y, et al., Intern Med, 2018; Shishido‐Hara Y and Kanoto M, Brain Nerve, 2020).

The biopsy should target an active site where JC virus proliferation occurs. Potential targets include the leading edge of expanding lesions, the interface between T1 hypointense regions, and areas of contrast enhancement on MRI. Additionally, the hyperintense rim observed on diffusion‐weighted imaging (DWI) represents a viable biopsy target. Although FLAIR images sensitively describe gliosis and histiocytic infiltration, they may not always indicate an active PML lesion. MRI is instrumental in revealing the localization and dynamics of expanding PML lesions, ²⁴ , ²⁵ , ²⁶ but careful interpretation is necessary.

Early cytopathology of JC virus‐infected cells

The detection of JC virus‐infected oligodendrocytes is critical for a definitive pathological diagnosis. Typically, JC virus‐infected cells exhibit enlarged, round nuclei containing amphophilic viral inclusions when stained with hematoxylin and eosin (HE). The JC viral antigen is detectable throughout the entire nucleus (full inclusion) in immunohistochemistry. However, in the early stages of infection, the characteristic JC virus‐infected cells may not be present due to a low viral copy number, and the nuclei of infected cells would be relatively small (Figure 3c). Recent studies have shown that the JC virus replicates within a specific intranuclear domain, promyelocytic leukemia bodies (PML‐NBs). Consequently, JC virus progeny accumulate in PML‐NBs during the early stages, forming dot‐shaped inclusions. ²⁷ , ²⁸ , ²⁹ , ³⁰ , ³¹ , ³²

Figure 3.

Cytopathology of JC virus‐infected Glial cells. (a) Nuclear enlargement in JC virus‐infected oligodendrocytes. Normal oligodendrocytes in the G0 resting stage exhibit small nuclei with compact chromatin. Upon viral infection, the nuclei enlarge as the cell cycle progresses from S to G2, during which JC virus progeny proliferate within developing PML‐NBs, Consequently, dot‐shaped inclusions appear before the formation of full inclusions. (b) In infected oligodendrocytes, Olig‐2 expression diminishes with nuclear enlargement, while Ki‐67/MIB‐1 expression becomes positive. In situ hybridization (ISH) for JC viral DNA detection is effective for early PML diagnosis, whereas immunohistochemistry (IHC) for JC viral protein detection lacks sensitivity (Shishido‐Hara et al., J Neuropathol Exp Neurol, 2014; Ishii J et al., Intern Med, 2018). (c) Histopathology of an early PML lesion. JC virus‐infected glial cells (indicated by yellow arrows) exhibit relatively small nuclei, surrounded by lymphocytes, plasma cells, and macrophages. (Nishiyama et al., Neurol Neuroimmunol Neuroinflamm, 2018). PML‐NBs, promyelocytic leukemia bodies.

Normal oligodendrocytes possess small, round nuclei with compact chromatin when in the G0 resting state. JC virus‐infected cells, however, activate the cell cycle from the S to the G2 phase, during which PML‐NBs enlarge. Within these expanding PML‐NBs, the JC virus replicates and forms dot‐shaped inclusions in the middle‐to‐large‐sized nuclei (Figure 3a). ³⁰ Immunohistochemically, these infected cells are positive for Ki‐67/MIB‐1. Although cells with enlarging nuclei are likely oligodendrocytes, specific biomarkers are lacking. Olig‐2 becomes negative as the nuclei enlarge, but confirming the negativity of GFAP and CD45 (LCA) is advisable to distinguish them from astrocytes and lymphocytes. Detection of JC virus‐infected cells using antibodies against capsid proteins, VP1 or VP2/VP3, is notably insensitive (Figure 3b). Even if a few positive cells are identified, their pathological significance may be difficult to assess. In situ hybridization (ISH) for JC virus DNA offers greater sensitivity; there is a case report in which JC virus‐infected cells were undetectable by immunohistochemistry, but more than 20 cells were identified as positive by ISH, leading to a final diagnosis of PML. ¹¹ PCR can also detect JC virus DNA in brain tissue obtained via biopsy. Although this is the most sensitive method for confirming JC virus infection, its presence, even in unaffected brain tissue, necessitates cautious interpretation.

Evaluation of demyelinating lesions in biopsy samples

While Kluver‐Barrera (KB) staining is commonly employed to assess demyelination in neuropathology, its interpretation can be difficult when only small brain tissue samples contain affected white matter. In such cases, evaluating infiltrating M2 macrophages through CD68 or CD163 immunohistochemistry is highly beneficial (Figure 1d–f). Additionally, staining for Olig2 to assess the proliferation of reactive oligodendrocytes is helpful in evaluating the early PML lesions.

Evaluation of inflammatory reactions

A systematic review of 47 papers for 52 cases of natalizumab‐associated PML between 2005 and 2016 indicated that approximately 80% of the cases exhibited contrast enhancement on MRI. ²² This reflects the presence of antiviral host inflammatory responses and usually consists of T, B, and plasma cells in brain tissues obtained via biopsy. Macrophages also infiltrate in demyelinating areas. A host inflammatory response suggests a better prognosis due to suppressed viral proliferation. However, the degree of inflammation varies across cases. In most instances, inflammatory reactions are mild, with a significantly low viral copy number. ¹² , ¹³ , ¹⁴ Nonetheless, in some cases, severe inflammatory responses have been observed, with pathological features resembling malignant lymphoma, potentially leading to misdiagnosis ¹⁵ , ¹⁶ Fortunately, high‐dose methotrexate (MTX) therapy has proven effective in reducing inflammation. ¹⁵ , ¹⁶ Therefore, when a biopsy is performed, it is crucial to evaluate the host inflammatory response qualitatively and quantitatively to assess the patient's physiological condition accurately (Figure 4).

Figure 4.

Anti‐JC virus response and the proposal of therapeutic strategies. The patient's physiological condition is likely determined by the balance between the host's inflammatory response and the viral load. (a) When host immunity is severely suppressed, antiviral therapy should be considered. (b,c) When host immunity is active, immune checkpoint inhibitors (ICIs), such as pembrolizumab, may be effective. (d,e) If the host immune response remains excessively active even after viral clearance, anti‐inflammatory strategies, such as high‐dose methotrexate (MTX) therapy, should be considered (Shishido‐Hara et al., Neuropathology, 2024).

T cell immunity

In cases of PML with moderate inflammatory reactions, diagnosis may be difficult due to the absence of characteristic JC virus‐infected cells. However, given the low viral load, the prognosis is generally favorable. For example, in the case reported by Aly et al. 2011, pathological investigation first revealed the perivascular and parenchymal lymphocyte infiltration associated with reactive gliosis and abundant foamy macrophages. The proportion of CD4⁺ cells was 70.4%, and that of CD8⁺ cells was 24.1%, leading to an initial diagnosis of inflammatory demyelinating disease. However, the JC virus was subsequently detected by ISH, resulting in a diagnosis of CD4⁺‐dominant PML‐IRIS. ³³ Similar cases of PML with T cell response were reported from Japan, with variability in the CD4⁺/CD8⁺ ratio among cases. ³⁴ Usually, the number of CD4⁺ T cells exceeds that of CD8⁺ T cells, with the CD4⁺/CD8⁺ ratio in three example cases being 1.41, 2.3, and 4.0, respectively. ¹² , ¹⁵ , ¹⁷ Only one case exhibited a predominance of CD8⁺ T cell infiltration over CD4⁺, and this case was resistant to steroid therapy (unpublished data).

B cell and plasma cell immunity

In PML with inflammatory reactions, plasma cell infiltration is frequently seen around the JC virus‐infected cells. CD20⁺ B cells typically accumulate as a perivascular cuff within the perivascular area. In contrast, CD79⁺ B cells and CD138⁺ plasma cells are usually found in these cuffs' outer regions or diffusely infiltrate the parenchyma. It is intriguing to consider that these perivascular cuffs might resemble lymphoid follicles involved in B cell maturation. In cases of PML with severe inflammation, plasma cell activation is evident, as indicated by the presence of binucleated plasma cells. ¹⁵ , ¹⁶ Thus, B‐cell and plasma cell immunity also play a role in the host's response against the JC virus.

What is fatal PML‐IRIS?

Immune reconstitution inflammatory syndrome (IRIS) is clinically defined as a “paradoxical deterioration” occurring in the context of host immune reconstitution. When a restored immune system recognizes JC virus antigens, this condition is termed PML‐IRIS. PML‐IRIS was first identified in AIDS patients undergoing antiretroviral therapy (ART), where the recovery of CD4⁺ cells led to an overwhelming immune response against JC virus antigens, often resulting in death. Similar physiological reactions have been observed in other clinical contexts, such as following blood cell count recovery after bone marrow transplantation or the discontinuation of immunosuppressive medications. The prognosis for PML‐IRIS was poor in AIDS patients receiving ART in the 1990s, but a more favorable outcome has been noted in drug‐associated PML cases since 2005. Consequently, there has been an ongoing debate as to whether inflammatory reactions in PML indicate a favorable prognosis or a poor outcome. ³⁵

Diagnostic criteria for PML‐IRIS have not yet been established based on pathological or immunological evidence. Clinically, PML‐IRIS was typically diagnosed by rapidly enlarging lesions with contrast enhancement on MRI. However, chronic low‐grade inflammation can persist even after the initial resolution of PML‐IRIS, indicating varying levels of host inflammatory response. However, co‐relating pathology has not yet been elucidated.

When a brain biopsy is performed from contrast‐enhanced lesions observed on MRI, pathology reveals an antiviral host inflammatory response at varying levels (Figure 4). In most cases, the inflammatory response is polymorphic, involving CD4⁺ and CD8⁺ T cells, B cells, and plasma cells. The prognosis is usually favorable. In contrast, autopsy cases of PML‐IRIS often report an excessive CD8⁺ T cell response. ³⁶ , ³⁷ A fatal case of PML‐IRIS occurred in an MS patient treated with natalizumab, leading to rapid clinical deterioration and death. Autopsy findings were similar to those seen in PML‐IRIS in AIDS patients receiving ART, characterized by a monomorphic immune response dominated by CD8⁺ T lymphocytes, with minimal or absent CD4⁺ T cells, B cells, and plasma cells. ³⁶ , ³⁷ , ³⁸ Notably, monoclonal T cell expansion was detected in the bone marrow and spleen. Therefore, the fatality of PML‐IRIS is likely associated with monomorphic CD8⁺ T cell response in the absence of CD4⁺.

The regulation of the antiviral immune response remains unclear, but the PD‐1/PD‐L1 immune checkpoint system likely plays a crucial role. Pathological investigations have shown that in PML with inflammatory reactions, T cells express PD‐1, while macrophages express PD‐L1. Pembrolizumab, a PD‐1 inhibitor, has been reported as effective in many PML cases. Therefore, the presence of a functioning immune checkpoint system may rapidly resolve inflammation once the host immune response clears the virus, potentially serving as a key determinant of prognosis. Although ICIs would be effective in clearing the virus, dysfunction of an immune checkpoint system with ICIs may lead to a new adverse event characterized by a monomorphic CD8⁺ T cell response.

DISCUSSION

This review explored the PML pathology frequently observed in brain tissues through biopsy. Particularly in cases of drug‐associated PML, brain biopsy is crucial due to the often low copy number of the JC viral genome in CSF, which may not be detectable by PCR. However, the diagnostic utility of biopsies would also be limited due to difficulty in recognizing JC virus‐infected cells. Moreover, inflammatory reactions are frequently seen, and interpretation is important to evaluate a patient's physiological conditions to determine the therapeutic strategies.

ICIs have recently emerged as a therapeutic option for PML. Pembrolizumab, a monoclonal antibody targeting PD‐1, is especially well‐documented. Some studies have documented clinical improvement, stabilization, or reductions in CSF viral load following pembrolizumab administration. ³⁹ , ⁴⁰ , ⁴¹ The efficacy of pembrolizumab may depend on factors such as early intervention and the patient's underlying health condition. ⁴² Although pembrolizumab has been employed in various immunocompromised populations, including those with HIV/AIDS, primary immunodeficiencies, and hematologic malignancies, the effectiveness is not universal. Adverse events have also been reported, and I propose that the evaluation of inflammatory cells in brain tissues obtained by biopsy is crucial to identify predictors of therapeutic response and optimize treatment protocols for PML.

CONCLUSION

Early pathological findings of PML, seen with brain biopsy, are different from those observed in autopsy cases in the final stage. In drug‐associated PML, the anti‐viral host response of polymorphic inflammatory cells may function at a low level, expecting the effectiveness of ICIs. However, dysregulation of the immune system may result in unfavorable outcomes. Evaluation of inflammatory cells would be helpful to optimize treatment strategies and ultimately enhance the prognosis for patients with PML.

AUTHOR CONTRIBUTIONS

YukikoShishido‐Hara contributed all to this publication, including the design of the manuscript, figure preparation, and manuscript writings.

CONFLICT OF INTEREST STATEMENT

Yukiko Shishido‐Hara (Y. S.‐H.) is an Editorial Board member of Pathology International and an author of this article. To minimize bias, Y. S.‐H. was excluded from all editorial decision‐making related to the acceptance of this article for publication.

Shishido‐Hara Y. Brain biopsy and pathological diagnosis for drug‐associated progressive multifocal leukoencephalopathy (PML) with inflammatory reactions. Pathol Int. 2024;74:673–681. 10.1111/pin.13492